1. Field of the Invention
This invention relates to cell growth substrates that possess improved properties with regard to the attachment and growth of cells. The invention relates to the production of novel cell growth substrates from materials that possess suitable bulk properties, but inappropriate surface chemical composition for good attachment and growth of cells. In a particular aspect the materials and method of this invention are useful for the fabrication of substrates for attachment and growth of human cell lines.
Good attachment and growth of cells on the surfaces of an artificial implant are critical for applications such as vascular grafts. However, human cells generally show little tendency to attach and grow evenly on the surface of articles made from polymeric materials. To overcome this deficiency, a number of modifications to the bulk polymer or the polymer surface have been described in the art. The synthesis of bulk polymers is one approach; another is the surface modification of known polymers to provide chemical groups that confer improved performance on the polymer surface. It is important to be able to optimize both the bulk properties and the surface properties with some degree of independence, and surface modification procedures are well suited to this aim.
2. Description of the Related Art
The interaction between a polymeric material and biomedical constituents such as proteins and cells occurs at the polymer surface. The interfacial forces are very short range, and therefore are determined by the chemical constituents at the surface and in the sub-surface region of the polymeric material, that is, chemical groups located within only a few molecular layers of the surface of the polymeric material. Surface modification techniques are thus ideally suited to the control of polymer properties governed by interfacial interactions, such as for biomedical applications. The growth of human cells on tissue culture dished is strongly dependent on the chemical composition of the polymer surface (Griesser, Johnson and Steele, Polymeric Materials Science and Engineering, in press), and it is presumed that the growth of endothelial cells inside a vascular graft following implantation is likewise affected by the composition of the polymer surface of the graft. A number of surface treatment techniques for polymeric materials are known in the art: Corona Discharge, Flame Treatment, Acid Etching, and a number of other methods intended to perform chemical modification of the surface. Disadvantages of these techniques comprise the use of or production of hazardous chemicals, the often excessive depth of treatment, non-uniformity of treatment at a microscopic level, and often severe etching and pitting that leads to changes in surface topography. The depth of treatment is important because with thin polymeric materials such as those required for small diameter vascular grafts the bulk properties soon become affected by an excessive treatment.
Treatment of polymeric surfaces by gas plasmas presents the advantages of very low treatment depth and uniformity on a microscopic scale. A gas plasma (also known as glow discharge) is produced by electrical discharge in a gas atmosphere at reduced pressure ("vacuum"). It creates a stable, partially ionized gas that may be utilized for effecting reactions on the surface of the polymer substrate because the gas plasma environment activates even chemical compounds that are unreactive under normal conditions. The treatment intensity at the surface is generally relatively strong, and yet, the penetration depth of gas plasma treatment is very low, of the order of 5 to 50 nanometres, at a treatment level sufficient for useful surface modification. Surface topography does not change unless exposure to the plasma is performed for extended periods of time usually much exceeding the time required for achieving the desired chemical modification. There occurs, therefore, much less alteration of the properties of the bulk polymer than with alternative treatment technologies.
A glow discharge in an oxygen containing atmosphere has been claimed to render polymeric surfaces more hydrophilic (U.S. Pat. No. 4,452,679). The treatment leads to formation of polar, oxygen containing functionalities on the surface. The main disadvantage of this approach is that the treatment is not permanent due to reorientation of the polymer chains with time because polymeric materials undergo unavoidable, thermally driven chain segmental motions. Wettability by water decreases as the hydrophilic surface groups produced by treatment become buried inside the polymer, as described by H. Yasuda et. al. in J. Polym. Sci.: Polym. Phys. Ed., 19 1285 (1981). The hydrophilic nature of the surface thus diminishes with storage time. Another disadvantage is that, such surfaces are not good substrates for cell attachment and growth per se, but they require the provision of an adhesion promoting protein prior to cell attachment and growth. An example of this is the surface modification of polystyrene, which has enabled the successful fabrication of cell culture dishes ("tissue culture polystyrene", TCP) which support good attachment and growth of human cells, but only if the dishes are precoated with adhesive protein such as fibronectin (C. Klein-Soyer, S. Hemmendinger and J. P. Cazenave, Biomaterials 1989, Vol. 10, 85). In the absence of pre-coating, cells grown on top look patchy and irregular.
Other hydrophilic surface modification procedures have been utilized in order to improve the attachment and growth of cells on tissue culture dishes and vascular grafts and the like articles where performance is dependent on good cell anchorage. The modification of surfaces in an ammonia plasma has been described and seems to provide cell compatible surfaces. However, chemical groups attached to the surface in this way are subject to the same thermally driven reorientation, which means that the newly created surface groups are slowly lost from the surface. Surface modification techniques are needed that provide permanent surface compositions and good cell response.
Tissue culture dishes provide a convenient means of studying the process of attachment and growth of cells, and it is considered that the surface treatment procedures suitable for the optimization of cell culture dishes are also suitable for the optimization of surgical implant materials. One cell growth substrate is available (Falcon Primaria tissue culture plate) which does not require pre-coating with absorbed adhesive protein. The reason for this is stated to be the incorporation of amine and amide groups into the polystyrene chain. Primaria does, however, suffer from the disadvantage that the adhesion of the extracellular matrix to the polymer surface is not strong, and that repair of lesions is slower that on other TCP dishes.
In spite of the considerable literature on cell growth substrates, the problem of providing stable surfaces on polymeric articles such that human cells attach and grow well without requiring the step of pre-coating with adhesive protein, the extracellular matrix adheres well, and cells are able to readily execute repair to any damage, has not yet been solved.